Predictive and prognostic biomarkers related to anti-angiogenic therapy of metastatic colorectal cancer

ABSTRACT

The present invention provides methods for treating metastatic cancer comprising identifying subjects who will respond favorably to anti-VEGF therapy. According to certain aspects of the invention, subjects are identified based on their expression level of one or more predictive biomarkers. Favorable response to anti-VEGF therapy is indicated by high expression levels of certain biomarkers or by low expression levels of certain biomarkers. An exemplary predictive biomarker is VEGF-A. Also disclosed herein are prognostic biomarkers useful for identifying cancer-bearing subjects who are expected to have better relative survival outcomes.

FIELD OF THE INVENTION

The present invention relates to the use of predictive and prognosticbiomarkers in identification and treatment of metastatic cancer inpatients.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a cytokine involved inangiogenesis. The ligand VEGF-A interacts with VEGF receptor-1 (VEGFR1)and VEGFR2, thereby initiating an angiogenesis signaling pathway innormal and tumor vasculature. Antagonists of VEGF are known to be usefulfor the treatment of a variety of diseases and disorders includingcancers, eye diseases and other conditions involving excessive, unwantedor inappropriate angiogenesis. An example of a VEGF antagonist isaflibercept (also known as VEGF Trap; or ziv-aflibercept, which ismarketed as ZALTRAP®, Regeneron Pharmaceuticals, Inc., Tarrytown, NY).Aflibercept is a VEGF receptor-based chimeric molecule comprising domain2 from VEGFR1 fused to domain 3 from VEGFR2, which is, in turn, attachedthrough the hinge region to the Fc(a) domain of human IgG1.Ziv-aflibercept is approved for the treatment of metastatic colorectalcancer and is being developed for the treatment of other cancerousconditions as well. VEGF Trap is described, e.g., in U.S. Pat. No.7,070,959; see also, Holash et al., Proc. Natl. Acad. Sci. USA99:11393-11398 (2002).

To date, no predictive biomarkers of resistance or susceptibility toVEGF inhibition have been validated. Although VEGF antagonists haveshown great promise in the treatment of cancer, validated biomarkersthat predict the efficacy of anti-VEGF therapy are needed for theeffective identification and selection of patient sub-populations thatrespond favorably to anti-VEGF therapy. Accordingly, an unmet needexists in the art for identifying and validating predictive andprognostic biomarkers in patients with metastatic cancer who areadministered anti-VEGF therapy.

BRIEF SUMMARY OF THE INVENTION

According to one aspect of the present invention, methods are providedfor treating cancer in a subject. For example, the methods according tothis aspect of the invention comprise administering a VEGF antagonist tothe subject, wherein the subject has been diagnosed with metastaticcancer and has been selected for treatment with a VEGF antagonist on thebasis of exhibiting elevated expression of a predictive biomarker suchas VEGF-A. In certain embodiments, the elevated expression of thepredictive biomarker is determined based on a comparison to lower levelof biomarker expression in cancer-bearing subjects. In one embodiment,the metastatic cancer is metastatic colorectal cancer (mCRC).

According to another aspect of the present invention, methods areprovided for treating advanced ovarian cancer in a subject. The methodsaccording to this aspect of the invention comprise administering a VEGFantagonist to the subject, wherein the subject has been diagnosed withadvanced ovarian cancer and has been selected for treatment with theVEGF antagonist on the basis of exhibiting variant (i.e., increased ordecreased) expression of interleukin-6 (IL-6). In certain embodiments,variant expression of IL-6 in the subject is determined based on acomparison to the level of IL-6 expression in non-cancer-bearingsubjects. Under circumstances in which a cancer patient is identified asexhibiting increased expression of IL-6, the patient may be effectivelytreated according to the present invention by administration of acombination of a VEGF antagonist and an IL-6 or IL-6 receptorantagonist.

According to another aspect of the present invention, methods areprovided for identifying a subject with metastatic colorectal cancer whois likely to respond favorably to anti-VEGF therapy. The methodsaccording to this aspect of the invention comprise obtaining a samplefrom the patient and measuring in the sample the level of a predictivebiomarker such as VEGF-A (or other predictive biomarker(s) as describedherein), wherein an elevated expression of the predictive biomarker ascompared to the lower level of biomarker expression (identified as“high” level as disclosed elsewhere herein) in a patient with mCRC,identifies the patient as a patient who is likely to respond favorablyto anti-VEGF therapy.

In some embodiments, the anti-VEGF therapy comprises administration of aVEGF antagonist to a subject in need thereof. The VEGF antagonist usedwith the methods of the present invention may be an anti-VEGF antibody,an anti-VEGF receptor antibody or a VEGF receptor-based chimericmolecule (VEGF Trap). In certain embodiments, the VEGF antagonist isziv-aflibercept.

In some embodiments, the anti-VEGF therapy may be administered incombination with a chemotherapeutic agent and/or regimen. Exemplarychemotherapeutic agents/regimens include folinic acid, 5-fluorouraciland oxaliplatin (i.e., the FOLFOX treatment), and folinic acid,5-fluorouracil and irinotecan (i.e., the FOLFIRI treatment).

In certain embodiments, the sample obtained from the patient is selectedfrom the group consisting of blood, serum and plasma.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of hazard ratio (HR) changes of the “high” biomarkergroup(s). The biomarker “high” group was defined by cutoff valuesranging from 88 to 1140 pg/mL of plasma VEGF biomarker concentrations ina subset of patients with metastatic colorectal cancer (mCRC) in a phase3 study of mCRC patients treated with aflibercept in combination withFOLFIRI.

FIG. 2 is a graph of HR changes of the “high” biomarker group(s). Thebiomarker “high” group was defined by cutoff values ranging from 5.1 to111 pg/mL of plasma IL-8 biomarker concentrations (in pg/mL) in a subsetof patients with mCRC in a phase 3 study of mCRC patients treated withaflibercept in combination with FOLFIRI.

FIG. 3 shows correlation of high or low interleukin-6 (IL-6) levels withthe overall surviving fraction of patients having ovarian cancer in aphase 2 study of advanced ovarian cancer patients treated withaflibercept monotherapy.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to describe intheir entirety.

Predictive and Prognostic Biomarkers

The present invention is based in part on the identification of certainbiomarker proteins whose expression levels (higher or lower than medianexpression levels) in cancer patients were found to correlate withenhanced overall survival (OS) following treatment with ananti-angiogenic agent. In particular, certain protein biomarkers wereidentified that when expressed at higher or lower levels in patientswith metastatic colorectal cancer (mCRC) correlated with enhancedoverall survival of such patients following treatment with the VEGFantagonist ziv-aflibercept. An exemplary biomarker that can be used inthe context of the present invention to identify patients likely torespond favorably to ziv-aflibercept treatment is VEGF-A. In certainembodiments, patients with higher levels of VEGF-A as compared to thelevel of VEGF-A expression in patients with mCRC and not treated with aVEGF antagonist, were found to have improved survival outcomes whentreated with anti-VEGF therapy. In certain embodiments, increased ordecreased expression of one or more biomarkers may be used as acomponent of a signature to identify patients likely to respondfavorably to ziv-aflibercept treatment. For example, the signature mayinclude VEGF pathway proteins (such as VEGF-R2 and VEGF-R3) orinflammation-related markers such as macrophage migration inhibitoryfactor (MIF) and surfactant protein D (SPD).

In a related aspect, the present invention relates to identification ofcertain protein biomarkers that when expressed at higher or lower thanmedian levels in patients with advanced ovarian cancer correlated withenhanced overall survival of such patients following treatment with theVEGF antagonist ziv-aflibercept. An exemplary biomarker that can be usedin the context of the present invention to identify patients likely torespond favorably to ziv-aflibercept treatment is the cytokineinterleukin-6 (IL-6). In certain embodiments, patients with lower levelsof IL-6 as compared to median level of IL-6 expression in patients withadvanced ovarian cancer and not treated with a VEGF antagonist, werefound to have improved survival outcomes when treated with anti-VEGFtherapy.

As used herein, a “predictive” biomarker refers to a biomarker thatgives information on the effect of a therapeutic intervention in apatient. The predictive biomarker may also be a target for therapy. Apredictive biomarker can be used for patient selection, specifically,for identifying patients that will respond favorably to a particulartherapy. In the context of the present invention, a predictive biomarkerincludes a protein biomarker with an elevated or reduced expression thatcorrelates with improved survival outcome of a patient having cancer(such as metastatic colorectal cancer or ovarian cancer) and treatedwith anti-VEGF therapy as compared to a similarly situated patient nottreated with anti-VEGF therapy. An example of a predictive biomarker,found in the study exemplified herein, is VEGF-A. A mCRC patient who hadelevated expression of VEGF-A and was treated with ziv-aflibercept had ahigher probability of overall survival as compared to a patient havingmCRC but not treated with ziv-aflibercept. Another example of apredictive marker is IL-6. A patient with ovarian cancer who hadelevated expression of IL-6 and was treated with ziv-aflibercept had alower probability of overall survival as compared to a patient havingovarian cancer but not treated with ziv-aflibercept. Additional examplesof predictive biomarkers include VEGF-R2, VEGF-R3, SPD and MIF. Certainbiomarkers may be categorized as both predictive and prognostic. Anexample of a biomarker that may be classified as both predictive andprognostic in the context of cancer treatment with a VEGF antagonist isIL-8.

The present invention also relates to the identification of certainprognostic biomarkers that correlate with mCRC overall survival.Prognostic biomarkers include proteins that when expressed at higher orlower than normal levels in patients with metastatic colorectal cancer(mCRC) correlate with enhanced overall survival of the patientsirrespective of treatment. Exemplary prognostic biomarkers that can beused in the context of the present invention to identify patients withpotentially enhanced mCRC overall survival include angiopoietin-2(Ang-2), C-reactive protein (CRP) and NRP1.

As used herein, a “prognostic” biomarker refers to a biomarker thatprovides information about a patient's overall cancer outcome,regardless of treatment. A prognostic biomarker may give information onrecurrence in patients who receive curative treatment. In someembodiments, altered expression of a prognostic biomarker correlateswith progression-free survival in a patient with metastatic disease. Inthe context of the present invention, the term “prognostic” biomarkerrefers to altered expression of a protein biomarker that correlates withpoor prognosis of metastatic cancer in a patient. For example, it isshown herein that mCRC patients with high plasma Ang-2 or CRP levels hadpoor prognosis of mCRC (e.g., decreased probability of survival)compared to patients with low Ang-2 or CRP levels, whether the patientswere treated with anti-VEGF therapy or not.

The level of predictive and prognostic biomarkers may be determined in apatient by directly or indirectly measuring the absolute or relativeamount of the biomarker in a tissue sample, tumor sample (e.g., biopsy),or fluid sample obtained from the patient. The fluid sample may beselected from the group consisting of blood, plasma and serum. Theamount of a biomarker may be measured in a sample using techniques suchas enzyme linked immunosorbent assay (ELISA), or other protein detectionand analytic methods, as well as by measuring the amount of nucleic acid(e.g., mRNA) that encodes the protein biomarker. Assays that involvedirectly measuring or detecting specific protein biomarkers in a samplecan be accomplished using, e.g., antibodies or other antigen-bindingproteins specific for the biomarker. Such antibodies or antigen-bindingproteins can be labeled with a detectable compound such as a fluorophoreor radioactive compound. Thus, the present invention also includesantibodies and antigen-binding proteins that specifically bind any ofthe predictive or prognostic biomarkers described herein, as well askits and diagnostic methods comprising such antibodies and uses thereof.

The reference level of expression of a particular biomarker may bedetermined as a single value or a range of values which is/aredetermined based on the expression level of the biomarker measured, forinstance, in a population of healthy subjects or in a population ofsubjects in need of therapy. According to certain embodiments of thepresent invention, the reference level of expression of a biomarker isdetermined based on the expression level of the biomarker measured in apopulation of subjects in need of a ziv-aflibercept therapy. Theanalyzed population may be divided into percentiles based on themeasured level of expression of a particular biomarker. The referencelevel in some instances can be defined as the percentile that providesthe best separation between patients suffering from a cancer on whichthe treatment with ziv-aflibercept is substantially effective ascompared to patients suffering from a cancer for which treatment withziv-aflibercept is less or sub-optimally effective. The reference levelof expression of a particular biomarker may vary (i) according to thesize of the studied population, and (ii) depending on the method usedfor measuring the expression of the biomarker.

VEGF Antagonists

As used herein, the expression “VEGF antagonist” means any molecule thatblocks, reduces or interferes with the normal biological activity ofvascular endothelial growth factor (VEGF) or a VEGF receptor. VEGFantagonists include molecules which interfere with the interactionbetween VEGF and a natural VEGF receptor, e.g., molecules which bind toVEGF or a VEGF receptor and prevent or otherwise hinder the interactionbetween VEGF and a VEGF receptor. Specific exemplary VEGF antagonistsinclude anti-VEGF antibodies (e.g., bevacizumab [AVASTIN®]), anti-VEGFreceptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2antibodies, etc.), and VEGF receptor-based chimeric molecules (alsoreferred to herein as “VEGF-Traps”).

VEGF receptor-based chimeric molecules include chimeric polypeptideswhich comprise two or more immunoglobulin (1g)-like domains of a VEGFreceptor such as VEGFR1 (also referred to as Flt1) and/or VEGFR2 (alsoreferred to as Flk1 or KDR), and may also contain a multimerizing domain(e.g., an Fc domain which facilitates the multimerization [e.g.,dimerization] of two or more chimeric polypeptides). An exemplary VEGFreceptor-based chimeric molecule is a molecule referred to asVEGFR1R2-FcΔC1(a) (also known as aflibercept or ziv-aflibercept, andmarketed under the product name ZALTRAP®) which is encoded by thenucleic acid sequence of SEQ ID NO: 1. VEGFR1R2-FcΔC1(a) comprises threecomponents: (1) a VEGFR1 component comprising amino acids 27 to 129 ofSEQ ID NO: 2; (2) a VEGFR2 component comprising amino acids 130 to 231of SEQ ID NO: 2; and (3) a multimerization component (“FcΔC1(a)”)comprising amino acids 232 to 457 of SEQ ID NO: 2 (the C-terminal aminoacid of SEQ ID NO: 2 [i.e., K458] may or may not be included in the VEGFantagonist used in the methods of the invention; see e.g., U.S. Pat. No.7,396,664). Amino acids 1-26 of SEQ ID NO: 2 are the signal sequence.

Biomarkers and Methods of Treatment of Metastatic Cancer

The present invention includes methods for treating cancer in a subject,e.g., advanced cancer, metastatic cancer, etc. The methods according tothis aspect of the invention comprise administering a VEGF antagonist tothe subject, wherein the subject has been diagnosed with cancer (e.g.,metastatic colorectal cancer, advanced ovarian cancer) and has beenselected for treatment with the VEGF antagonist on the basis ofexhibiting elevated expression of a biomarker wherein the elevatedexpression of the biomarker is determined based on a comparison to thelower level of expression (i.e., reference level) of the respectivebiomarker in subjects with metastatic cancer. The VEGF antagonist maycomprise a VEGF receptor-based chimeric molecule (VEGF Trap). An exampleof a VEGF Trap is ziv-aflibercept. In certain embodiments, the patientmay be administered a VEGF antagonist in combination with achemotherapeutic regimen comprising leucovorin (also known as folinicacid), 5 fluorouracil and irinotecan (the combination of which isreferred to as “FOLFIRI”).

The terms “subject” and “patient” are used interchangeably herein andrefer to human subjects in need of treatment for a cancer, preferablymetastatic cancer.

The present invention also includes methods for identifying a patientwith metastatic colorectal cancer (mCRC) who is likely to respondfavorably to anti-VEGF therapy. The methods comprise obtaining a samplefrom the patient and measuring in the sample the level of a biomarkersuch as VEGF-A, VEGF-R2, VEGF-R3, MIF and/or IL-8, wherein the elevatedexpression of the biomarker as compared to the lower level of expression(i.e., reference level) of the biomarker in subjects with mCRC,identifies the patient as a patient who is likely to respond favorablyto anti-VEGF therapy. In alternate embodiments, the methods compriseobtaining a sample from the patient and measuring in the sample thelevel of a biomarker such as SPD, wherein lower expression of thebiomarker as compared to the higher level of expression (i.e., referencelevel) of the biomarker in subjects with mCRC, identifies the patient asa patient who is likely to respond favorably to anti-VEGF therapy.

The present invention also includes methods for identifying a patientwith advanced ovarian cancer who is likely to respond favorably toanti-VEGF therapy. The methods comprise obtaining a sample from thepatient and measuring in the sample the level of a biomarker such asIL-6, wherein the variant expression of the biomarker as compared to themedian level of expression (i.e., reference level) of the biomarker insubjects with ovarian cancer, identifies the patient as a patient who islikely to respond favorably to anti-VEGF therapy.

As used herein, a patient “likely to respond favorably” to anti-VEGFtherapy refers to a patient having a cancer (e.g., mCRC, ovarian cancer)who upon administration of said anti-VEGF therapy is expected to show aneffect selected from the group consisting of increased or improvedoverall survival as compared to a patient having the cancer and not onanti-VEGF therapy, increased progression-free survival, tumorregression, and decreased probability of tumor relapse. Improvedsurvival time means about 1 week, 2 week, 4 week, 2 month, 4 month, 6month, 8 month, 10 month, 12 month, 14 month, 16 month, 18 month, 20month, 22 month, 24 month, 26 month, 28 month, 30 month, 36 month, 40month, or longer survival as compared to similarly situated subjectswith the cancer who do not receive anti-VEGF therapy.

The present invention also includes methods of determining prognosis ofmCRC in a subject. The methods according to this aspect of the inventioncomprise obtaining a sample from the subject and measuring in the samplethe level of a biomarker, wherein variant expression of the biomarker(elevated or reduced expression) as compared to the median level ofexpression of the respective biomarker in subjects with mCRC, identifiesthe patient as a patient with poor prognosis. In certain embodiments,the biomarker is selected from the group consisting of angiopoietin-2(Ang-2) and C-reactive protein (CRP). In certain embodiments, reducedexpression of a biomarker identifies the patient as a patient with goodprognosis. In certain embodiments, good prognosis includes an effectselected from the group consisting of increased overall survival,progression-free survival, reduced tumor growth, tumor regression, andinhibition of tumor relapse in the patient. For example, in the presentstudy, the inventors discovered that low plasma levels of Ang-2 or CRPcorrelated with higher probability of overall survival of a patient withmCRC.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Identification of Potential Predictive and PrognosticBiomarkers in Baseline Plasma Samples from a Phase 3 Clinical Trial ofVEGF Trap in Patients with Metastatic Colorectal Cancer Introduction

ZALTRAP® (ziv-aflibercept, also known as VEGF Trap), a VEGF-R1-R2-Fcchimeric protein that binds and neutralizes ligands of VEGFR1 and VEGFR2(eg VEGF-A, PLGF, and VEGF-B), has been approved for the treatment ofmetastatic colorectal cancer (mCRC) (specifically in combination withFOLFIRI [a chemotherapeutic regimen comprising irinotecan,5-fluorouracil, and leucovorin] in patients who have progressed afterprevious therapy including oxaliplatin).

Plasma and tumor samples were analyzed from a 1226-patient pivotal,randomized and placebo controlled registration trial of FOLFIRI+/−ziv-aflibercept (VELOUR, ClinicalTrials.gov Identifier: NCT00561470),in an effort to identify both prognostic and predictive factors. ThisExample sets forth an analysis of the baseline plasma samples, with thegoal of identifying possible biomarkers of ziv-aflibercept response.

Results

A retrospective analysis of protein biomarkers from 553 baseline plasmasamples was carried out. The population represented by the samplescollected was similar to the overall VELOUR population. Samples wereanalyzed for levels of 98 analytes using multiplex immunoassays andELISA. Biomarker values were dichotomized to biomarker “high” and “low”groups, based on the median value and then analyzed with respect tooverall survival (OS) of the patient upon treatment.

For OS, the hazard ratio (HR) was 0.809 in the plasma biomarkerpopulation vs. HR=0.817 in the overall VELOUR population. Forprogression-free survival (PFS), the HR=0.752 in the plasma biomarkerpopulation vs. HR=0.758 in VELOUR. Patient demographics, including ECOGstatus, were similar between the groups. Several biomarkers wereidentified as potentially predictive or prognostic (or both) of OS, witha HR<0.7 (false discovery rate of 0.05 and interaction p<0.10). Nobiomarker subset corresponded to worse OS with ziv-aflibercepttreatment.

Eight biomarkers were identified as potentially predictive of OS (HazardRatio (HR)<0.7; p<0.01, and were significant even after accounting formultiple testing), while 23 biomarkers were identified as potentiallyprognostic of OS (p<0.01). High levels of VEGF-A (at median=142 pg/mL)emerged as one of the potential predictive biomarkers of response toziv-aflibercept therapy. Table 1 shows a primary analysis of the medianoverall survival of control and ziv-aflibercept-treated patients havinghigh or low plasma VEGF-A levels.

TABLE 1 Primary analysis of the median overall survival of control andziv- aflibercept-treated patients having high or low plasma VEGF-Alevels Median OS for Median OS for ziv- Log-rank Control (95% CI)aflibercept (95% CI) HR (95% CI) test p-value Total: Number of event,n/N (%) 207/265 (78.1%) 195/288 (67.7%) VEGF (Low level): Number of95/132 (72%) 96/142 (67.6%) event, n/N (%) VEGF _(Low level) 13.1 (10.8to 17.1) 12.8 (11.9 to 16.2) 0.963 (0.725 to 1.28) 0.7947 VEGF (Highlevel): Number of 112/133 (84.2%) 99/146 (67.8%) event, n/N (%) VEGF_(High level) 9.7 (8.5 to 11.3) 12.5 (10.4 to 15.9) 0.644 (0.49 to0.845) 0.0013

As shown in Table 1, patients with high plasma VEGF-A levels that weretreated with ziv-aflibercept showed higher overall survival than controlgroup. HR was calculated to be 0.644 (interaction p-value: 0.056) whichindicated VEGF-A to be a potential predictive marker. Comparable resultswere shown with respect to PFS (HR=0.599, 95% Cl: 0.453-0.792, p=0.001).Similarly, overall response rate (ORR) increased from 6.2% in thecontrol group to 22.7% in the aflibercept-treated group in patients withhigh VEGF-A levels.

VEGF values within a range of 88-1140 pg/mL (categorized as “high”) wereused to determine cutoff values at 5% step-up increments. HR for OS wasstable with VEGF cutoff level <222 pg/mL (FIG. 1). At higher VEGF levels(>335 pg/mL), the ziv-aflibercept effect was not as pronounced (highest20% of patients).

VEGF pathway proteins VEGF-R2 and VEGF-R3 were identified as potentialpredictive biomarkers. Patients with high plasma sVEGF-R2 (median: 4.2pg/mL) who were treated with ziv-aflibercept were potentially found tohave better OS than control group (HR=0.686, 95% Cl: 0.49-0.85,p=0.008). Comparable results were found with PFS (HR=0.679, 95% Cl:0.516-0.893, p=0.005). Patients with high plasma sVEGF-R3 (median: 35pg/mL) who were treated with ziv-aflibercept were potentially found tohave better OS than control group (HR=0.686; p=0.006). Comparableresults were obtained for PFS (HR=0.711, 95% Cl: 0.545-0.927, p=0.01).

Surfactant protein D (SPD) was identified as a potential predictivemarker. Patients with low plasma SPD levels that were treated withziv-aflibercept showed higher overall survival than control group (13.9months as compared to 9.7 months; HR=0.598, 95% Cl: 0.453-0.791, p value<0.001). Comparable results were seen for PFS (HR=0.581, 95% Cl:0.437-0.773, p value <0.001). Overall response rate increased from8.1%in the control group to 25.6% in the aflibercept-treated group inpatients with low SPD levels.

Macrophage Migration Inhibitory Factor (MIF) was identified as apotential predictive marker. Patients with high plasma MIF that weretreated with ziv-aflibercept showed higher overall survival than controlgroup (12.7 months as compared to 9.5 months; HR=0.67, 95% Cl:0.512-0.875, p value: 0.003). Similar results were shown with PFS(HR=0.607, 95% Cl: 0.463-0.797, p value <0.001). Overall response rateincreased from 10.5%in the control group to 20.9% in theaflibercept-treated group in patients with high plasma MIF levels.

Eotaxin-1 (CCL11) was identified as a potential predictive marker.Patients with high plasma eotaxin-1 that were treated withziv-aflibercept showed higher probability of survival (HR: 0.661, 95%Cl: 0.497-0.879; p-value 0.004). Similar results were shown with PFS(HR=0.701, 95% Cl: 0.528-0.929, p value 0.012). Overall response rateincreased from 8.7% in the control group to 23.3% in theaflibercept-treated group in patients with high plasma eotaxin-1 levels.In multivariate analyses, eotaxin-1 was predictive of response toziv-aflibercept.

Hepsin was identified as a potential predictive marker. Patients withhigh plasma hepsin that were treated with ziv-aflibercept showed higherprobability of survival (HR: 0.671, 95% Cl: 0.511-0.881; p-value 0.004).Similar results were shown with PFS (HR=0.661, 95% Cl: 0.501-0.871, pvalue 0.003). Overall response rate increased from 10% in the controlgroup to 22.5% in the aflibercept-treated group in patients with highplasma hepsin levels. In multivariate analyses, hepsin was predictive ofresponse to ziv-aflibercept.

Some biomarkers were identified as potentially both predictive andprognostic. Interleukin-8 (IL-8) may be a predictive as well as aprognostic marker. Patients with low plasma IL-8 levels that weretreated with ziv-aflibercept showed higher overall survival (9.4 monthsas compared to 8.0 months; HR=0.632, 95% Cl: 0.489-0.817, p value:0.0006) than control-treated patients. Comparable results were obtainedfor PFS (4.9 months as compared to 3.9 months) with HR=0.694, 95% Cl:0.534-0.902, p value: 0.005). IL-8 was also identified as a potentialprognostic marker. Patients with low plasma IL-8 levels were found tohave better OS (18.8 months as compared to 9.4 months foraflibercept-treated patients, HR=2.319, p=0; and 19.8 months as comparedto 8.0 months for control, HR=4.48, p=0).

IL-8 values within a range of 5.1-111 pg/mL (categorized as “high”) wereused to determine cutoff values at 5% step-up increments. HR for OS wasstable with IL-8 cutoff level <49 pg/mL (FIG. 2). The best HR was seenat levels between 20-31 pg/mL. At higher IL-8 levels (>63 pg/mL), theziv-aflibercept effect was not as pronounced (highest 20% of patients).

Neuropilin-1 (NRP1) may be a prognostic marker. Patients with low plasmaNRP1 were found to have better OS for aflibercept-treated patients (18.7months as compared to 10.0 months, HR=2.104, p<0.001) and for control(14.2 months as compared to 9.0 months, HR=2.032, p<0.001).

Angiopoietin 2 (Ang-2) and C-reactive protein emerged as potentialprognostic markers.

Table 2 shows primary analysis of the median overall survival of controland treated patients having high or low levels of Ang-2.

TABLE 2 Primary analysis of the median overall survival of control andtreated patients having high or low levels of Ang-2 Median OS for MedianOS for ziv- Log-rank Control (95% CI) aflibercept (95% CI) HR (95% CI)test p-value Total: Number of event, n/N (%) 207/265 (78.1%) 195/288(67.7%) Ang-2 (Low level): Number of 85/118 (72%) 87/152 (57.2%) event,n/N (%) Ang-2 (Low level) 13.7 (11.7 to 17.7) 18 (14.4 to 21.8)  0.74(0.549 to 0.998) 0.0475 Ang-2 (High level): Number of 122/147 (83%)108/136 (79.4%) event, n/N (%) Ang-2 (High level) 9.6 (9 to 11.3) 10.3(8.5 to 12.2) 0.892 (0.687 to 1.159) 0.3894

As shown in Table 2, patients with low plasma Ang-2 levels (<3.9 ng/mL)showed better survival outcomes than patients with high Ang-2 levels,regardless of treatment. High levels of Ang-2 correlated with a poorprognostic effect both in control and treated patient subsets (HR=1.83in treated patients; HR=1.54 in control) (interaction p-value: 0.366).No statistically significant predictive effect was found to beassociated with Ang-2 expression.

Table 3 shows primary analysis of the median overall survival of controland treated patients having high or low levels of CRP.

TABLE 3 Primary analysis of the median overall survival of control andtreated patients having high or low levels of CRP Median OS for MedianOS for ziv- Log-rank Control (95% CI) aflibercept (95% CI) HR (95% CI)test p-value Total: Number of event, n/N (%) 207/265 (78.1%) 195/288(67.7%) CRP (Low level): Number of 85/131 (64.9%) 76/145 (52.4%) event,n/N (%) CRP _(Low level) 17.4 (13.7 to 20.5) 19.1 (16.7 to 25.1) 0.765(0.56 to 1.045)  0.0905 CRP (High level): Number of 122/134 (91%)119/143 (83.2%) event, n/N (%) CRP _(High level) 8.6 (7.1 to 9.7) 9.4(7.8 to 11.5) 0.763 (0.592 to 0.984) 0.0354

As shown in Table 3, patients with low plasma CRP levels (<9.4 μg/mL)showed better survival outcomes than patients with high CRP levels,regardless of treatment. High levels of CRP correlated with a poorprognostic effect both in control and treated patient subsets (HR=2.553in treated patients, p<0.001; and HR=2.773 in control, p<0.001). Nopredictive effect was found to be associated with CRP expression.

No biomarker subset corresponded to worse OS with ziv-aflibercepttreatment.

Table 4 summarizes the top potential predictive marker results foraflibercept treatment.

TABLE 4 Top potential predictive marker results in the study High or LowHazard bio- Ratio Inter- Bio- Median marker (ZT vs. action marker (min,max) group control) P-value* P-value IL-8 20 pg/mL High 0.63 4.00E−040.022 (2, 4504 pg/mL) MIF 0.3 ng/mL High 0.67 0.003  0.087 (0.015, 29ng/mL) Eotaxin-1 73 pg/mL High 0.66 0.0041 0.087 (27-487 pg/mL) VEGF 142pg/mL High 0.64 0.0013 0.056 (25, 2350 pg/mL) VEGFR2 4.2 pg/mL High 0.690.0082 0.157{circumflex over ( )} (1, 9 pg/mL) VEGFR3 35 ng/mL High 0.690.0061 0.177{circumflex over ( )} (3, 125 pg/mL) Hepsin 771 pg/mL High0.69 0.0038 0.06 (190-1860 pg/mL) SPD 7.7 ng/mL Low 0.60 3.00E−04 0.003(0.19, 85 ng/mL) *Log rank P-values were adjusted for false discoveryrate {circumflex over ( )}VEGF-R2 and VEGF-R3 fall below levels ofsignificance

TABLE 5 Potentially prognostic markers in the study Control treatedZiv-aflibercept-treated Hazard Hazard Ratio Ratio Bio- Median (High vs.(High vs. marker (min, max) Low) P-value Low) P-value IL-8 20 pg/mL4.4810 <0.001 2.3189 <0.001 (2, 4504 pg/mL) CRP 9.4 mg/mL 2.7732 <0.0012.5535 <0.001 (6, 390 pg/mL) NRP1 160 ng/mL 2.0324 <0.001 2.104 <0.001(34, 387 ng/mL) ANG2 3.9 ng/mL 1.5447 0.002 1.8293 <0.001 (1, 59 pg/mL)

Conclusions

In the present study, multiple predictive biomarkers of response toziv-aflibercept have been identified including e.g., VEGF-A. Patients inthe high VEGF-A group (i.e., plasma level >142 pg/mL) seemed to benefitthe most from aflibercept treatment. Similar results were observed withVEGF-R2 and VEGF-R3. VEGF-R2 and VEGF-R3 may be of interest for furtherstudy as component of a signature. However, at the highest VEGF levels,the benefit was found to decline. In addition, several prognosticbiomarkers were also identified in the course of the present analysis.Patient subsets with elevated expression of alternative angiogenicfactors (e.g., Ang-2) or increased inflammatory markers, (e.g., CRP)correlated with poor outcome. Low NRP-1 may be a prognostic marker forsurvival. Samples containing high VEGF pathway markers also correlatedwith poor outcome in the absence of ziv-aflibercept treatment.Identification of the relevant pathways in patients will be important tooptimize the efficacy of combination therapies.

Example 2: Identification of Potential Biomarkers in Serum Samples froma Phase 2 Clinical Trial of VEGF Trap in Patients with Ovarian Cancer

In an international, double-blind, phase 2 study of advanced ovariancancer (ClinicalTrials.gov Identifier: NCT00327171), aflibercept wasused as a monotherapy. Patients received either a 2 mg/kg or 4 mg/kgdose of aflibercept every two weeks and were monitored for overallresponse. Serum samples were analyzed to identify potential predictiveand prognostic markers. In this example, serum levels of interleukin-6(IL-6) were measured in a subset of 96 patients.

The study showed a modest response rate of 5% in both arms. When thedata was stratified based on low (<median) and high (>median) IL-6 serumlevels, it became apparent that patients with high IL-6 levels (>median)show a worse response (i.e., significantly poorer survival) compared tothose with low IL-6 levels (<median) (FIG. 3). The results suggest acorrelation between high levels of IL-6 and poorer response to anti-VEGFtherapy. Elevated IL-6 levels may be a direct or indirect cause ofresistance to anti-VEGF therapies observed in certain patients. Thus,the results of this Example suggest that screening cancer patients forelevated levels of IL-6 may identify certain patients whose therapeuticoutcome would be enhanced by combining anti-VEGF therapy with anti-IL-6or anti-IL-6R therapy, thereby counteracting or delaying the developmentof resistance to anti-VEGF therapies in these patients.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1.-7. (canceled)
 8. A method for identifying a patient with metastaticcolorectal cancer (mCRC) who is likely to respond favorably to VEGF TrapVECF therapy wherein the VEGF Trap comprises one or more immunoglobulin(Ig)-like domains of VEGFR1, one or more Ig-like domains of VEGFR2, anda multimerizing domain, the method comprising obtaining a sample fromthe patient and measuring in the sample the level of VEGF-A; wherein anelevated level of expression of VEGF-A, identifies the patient as apatient who is likely to respond favorably to anti-VEGF therapy, whereinthe elevated expression of VEGF-A is determined based on a comparison tothe lower level of VEGF-A in subjects with mCRC.
 9. The method of claim8, wherein the sample is blood, serum or plasma. 10.-11. (canceled) 12.The method of claim 9, wherein the VEGF Trap comprises Ig-like domain 2of VEGFR1, Ig-like domain 3 of VEGFR2, and a multimerizing domain. 13.The method of claim 9, wherein the VEGF Trap is ziv-aflibercept.
 14. Themethod of claim 13, wherein the expression level of VEGF-A is measuredusing an ELISA.
 15. The method of claim 13 wherein the VEGF Traptherapy, further comprising: administering to the subject achemotherapeutic regimen comprising irinotecan, 5-fluorouracil, andleucovorin (FOLFIRI).